Ruff sperm

Zebra finch sperm

Notes on the method

Sperm was collected in May/June 2021 from captive ruffs by abdominal massage (for a detailed protocol of sperm collection – including video - and sample preparation see [here]shorturl.at/ijnp9). Sperm (~0.5–3μl) was pipetted from the cloaca and immediately diluted and gently mixed in 50μl of preheated (40°C) Dulbecco’s Modified Eagle’s Medium (Advanced D-MEM, Invitrogen™). An aliquot of 2.5μl was pipetted onto a standard 20μm two-chamber count slide (Leja, The Netherlands) placed on a thermal plate (Tokai Hit, Tokai Hit Co., LtD.) kept at 40 °C. The slide was inspected and either recorded (if sperm densities were fine) or a new aliquot was taken and further diluted.

Sperm was recorded immediately after sample collection at 25 frames per second in eight different fields of the Leja slide under a 100x magnification using phase contrast and a digital camera (UI-1540-C, Olympus) mounted on a microscope (CX41, Olympus) fitted with a thermal plate (Tokai Hit, Tokai Hit Co., LtD.) kept at a constant temperature of 40°C.